Marine phospholipids are useful in medical products, health food and human nutrition, as well as in fish feed and means for increasing the rate of survival of fish larval and fry of marine species like cod, halibut and turbot.
Phospholipids from marine organisms comprise omega-3 fatty acids. Omega-3 fatty acids bound to marine phospholipids are assumed to have particularly useful properties.
Products such as fish milt and roe are traditional raw materials for marine phospholipids. However, these raw materials are available in limited volumes and the price of said raw materials is high.
Krill are small, shrimp-like animals, containing relatively high concentrations of phospholipids. In the group Euphasiids, there is more than 80 species, of which the Antarctic krill is one of these. The current greatest potential for commercial utilization is the Antarctic Euphausia superba. E. superba has a length of 2-6 cm. Another Antarctic krill species is E. crystallorphias. Meganyctiphanes norvegica, Thysanoessa inermis and T. raschii are examples of northern krill.
Fresh hill contains up to around 10% of lipids, of that approximately 50 of % phospholipids in Euphausia superba. Phospholipids from krill comprise a very high level of omega-3 fatty acids, whereof the content of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) is above 40%. The approximate composition of lipids from the two main species of Antarctic krill is given in Table 1.
TABLE 1Composition of krill lipids. Lipidclasses, (approximate sum EPA + DHA)RatioWax estersGlyceridesPhospholipidsEPA/DHAEuphausia150 (7)50 (40-50)1.4-1.5superbaEuphausia4020 (4)40 (30-40)1.3crystallorphias
Furthermore, Antarctic krill has lower level of environmental pollutants than traditional fish oils.
A typical composition of commercially available krill oil is as follows:
TABLE 2Fatty AcidA %C14:019C16:022C16:113C18:01C18:1n-9 +25C18:1n-7C18:2n-62C18:3n-31C20:12EPA4DHA1
A sample of the commercial product Superba™ Krill Oil (Aker Biomarine ASA, Norway) has been analyzed as having the following composition:
TABLE 3Fatty AcidA %C14:010C16:020C16:15C18:01C18:1n-9 +16C18:1n-7C18:2n-62C18:3n-32C18:4n-35EPA20DHA12
The krill has a digestive system with enzymes, including lipases that are very active around 0° C. The lipases stay active after the krill is dead, hydrolyzing part of the krill lipids. An unwanted effect of this is that krill oil normally contains several percents of free fatty acids. If the krill has to be cut into smaller fragments before being processed, the person skilled in the art will immediately realize that this will increase the degree of hydrolysis. Thus, it is a desire to find a process that can utilize whole, fresh krill, or whole body parts from krill, as such a process will provide a product with improved quality and low degree of hydrolysis of lipids. This improved quality will affect all groups of krill lipids, including phospholipids, triglycerides and astaxanthin esters.
Krill lipids are to a large extent located in the animals' head. A process that can utilize fresh krill is therefore also well suited for immediate processing of the by-products from krill wherefrom the head is peeled off, a product that can be produced onboard the fishing vessel.
From U.S. Pat. No. 6,800,299 of Beaudion et al. it is disclosed a method for extracting total lipid fractions from krill by successive extraction at low temperatures using organic solvents like acetone and ethanol. This process involves extraction with large amounts of organic solvents which is unfavorable.
K. Yamaguchi et al. (J. Agric. Food Chem. 1986 34, 904-907) showed that supercritical fluid extraction with carbon dioxide, which is the most common solvent for supercritical fluid extraction, of freeze dried Antarctic krill resulted in a product mainly consisting of non-polar lipids (mostly triglycerides), and no phospholipids. Yamaguchi et al. reported that oil in krill meal was deteriorated by oxidation or polymerization to such an extent that only limited extraction occurred with supercritical CO2.
Y. Tanaka and T. Ohkubo (J. Oleo. Sci. (2003), 52, 295-301) quotes the work of Yamaguci et al. in relation to their own work on extraction of lipids from salmon roe. In a more recent publication (Y. Tanaka et al. (2004), J. Oleo. Sci., 53, 417-424) the same authors try to solve this problem by using a mixture of ethanol and CO2 for extracting the phospholipids. By using CO2 with 5% ethanol no phospholipids were removed from freeze dried salmon roe, while by adding 10% ethanol, 30% of the phospholipids were removed, and by adding as much as 30% ethanol, more than 80% of the phospholipids were removed. Freeze drying is a costly and energy consuming process, and not suited for treatment of the very large volumes of raw materials that will become available by commercial krill fisheries.
Tanaka et al. tried to optimize the process by varying the temperature of the extraction, and found that low temperatures gave the best results. 33° C., a temperature just above the critical temperature for CO2, was chosen as giving best results.
Contrary to these findings, we have surprisingly found a process for extraction of a substantially total lipid fraction from fresh krill, without the need for complicated and costly pre-treatment like freeze drying of large volumes. The lipid fraction contained triglycerides, astaxanthin and phospholipids. We did not have to dry or deoil the raw material before processing. Contrary to Tanaka et al. we have found that a short heating of the marine raw material was positive for the extraction yield. It was also shown that pre-treatment like a short-time heating to moderate temperatures, or contact with a solid drying agent like molecular sieve, of the krill can make ethanol wash alone efficient in removing phospholipids from fresh krill. These findings have been the basis for the invention disclosed in International Patent Application No. PCT/NO2007/000402, which is incorporated herein by reference.
Now it has surprisingly been found that pre-treatment by microwaves on the hill raw material can be carried out before performing the process according to PCT/NO2007/000402. Microwave treatment is easily adaptable to frozen krill. When frozen krill is treated with microwaves of a suitable energy, thawing, or heating to moderate temperatures (10-30° C.) can be suitable in order to make ethanol wash alone sufficient in removing phospholipids from the hill. As described in the examples below, microwave treatment can release even more phospholipids from the hill material than heating without the use of microwaves.
The exposure to the fluid under supercritical pressure will prevent oxidation from taking place, and the combined carbon dioxide/ethanol is expected to deactivate any enzymatic hydrolysis of the hill lipids. As the process according to the invention requires a minimum of handling of the raw materials, and is well suited to be used on fresh krill, for example onboard the fishing vessel, the product according to the invention is expected to contain substantially less hydrolyzed and/or oxidized lipids than lipid produced by conventional processes. This also means that there is expected to be less deterioration of the krill lipid antioxidants than from conventional processing. The optional pre-treatment involving short-time heating of the fresh krill will also give an inactivation of enzymatic decomposition of the lipids, thus ensuring a product with very low levels of free fatty acids.
In International Patent Application No. PCT/NO2007/000402 it is provided a process for extracting a substantially total lipid fraction from fresh krill, comprising the steps of:
a) reducing the water content of krill raw material; and
b) isolating the lipid fraction.
Optionally, the above-mentioned process comprising a further step of:
a-1) extracting the water reduced krill material from step a) with CO2 at supercritical pressure containing ethanol, methanol, propanol or iso-propanol. This step, a-1), is performed directly after step a).
In one embodiment it is provided a process for extracting a substantially total lipid fraction from fresh krill, comprising the steps of:
a) reducing the water content of krill raw material;
a-1) extracting the water reduced krill material from step a) with CO2 containing ethanol, the extraction taking place at supercritical pressure; and
b) isolating the lipid fraction from the ethanol.
In a preferred embodiment, step a) comprises washing of the krill raw material with ethanol, methanol, propanol and/or iso-propanol in a weight ratio 1(krill):0.3(ethanol) to 1:5, more preferably 1:0.5 to 1:1. The washing may be performed using all the ethanol in one operation, or by dividing the total amount of ethanol between several sequential steps.
Preferably, the krill raw material is heated to 60-100° C., more preferred to 70-100° C., and most preferred to 80-95° C., before washing. Furthermore, the hill raw material is preferably heated for about 1 to 40 minutes, more preferred about 1 to 15 minutes, and most preferred for about 1 to 5 minutes, before washing.
In another preferred embodiment of the invention fresh or frozen hill are treated with microwaves before washing.
After washing, the alcohol will contain krill lipids, including a significant part of the krill phospholipids.
In another embodiment, step a) comprises bringing the hill raw material in contact with molecular sieve or another form of membrane, such as a water absorbing membrane or a water permeable membrane, for removal of water.
Preferably, the amount of ethanol, methanol, propanol and/or iso-propanol in step a-1) is 5-20% by weight, more preferably 10-15% by weight.
In addition to producing a product containing the total lipids of hill, phospholipids can be separated from the other lipids. To separate the total lipids obtained by extraction at supercritical pressure, according to the present invention into the different lipid classes, extraction of the said total lipids with pure carbon dioxide can remove the non-polar lipids from the omega-3 rich phospholipids. Extraction of the total lipids with carbon dioxide containing less than 5% ethanol or methanol is another option.
As the phospholipids are much richer in the valuable omega-3 fatty acids than the other lipid classes, high concentrates of omega-3 fatty acids can be produced. While commercially available fish oils comprise 11-33% total omega-3 fatty acids (Hjaltason, B and Haraldsson, G G (2006) Fish oils and lipids from marine sources, In: Modifying Lipids for Use in Food (F D Gunstone, ed), Woodhead Publishing Ltd, Cambridge, pp. 56-79), the phospholipids of hill contain much higher levels (Ellingsen, T E (1982) Biokjemiske studier over antarktisk hill, PhD thesis, Norges tekniske hoyskole, Trondheim. English summary in Publication no. 52 of the Norwegian Antarctic Research Expeditions (1976/77 and 1978/79)), see also Table 1.
The omega-3 rich phospholipids can be used as they are, giving the various positive biological effects that are attributed to omega-3 containing phospholipids. Alternatively, the phospholipids can be transesterified or hydrolyzed in order to give esters (typically ethyl esters) or free fatty acids or other derivatives that are suitable for further concentration of the omega-3 fatty acids. As examples, the ethyl esters of krill phospholipids will be valuable as an intermediate product for producing concentrates that comply with the European Pharmacopoeia monographs no. 1250 (Omega-3-acid ethyl ester 90), 2062 (Omega-3-acid ethyl esters 60) and 1352 (Omega-3-acid triglycerides). At the same time, the remaining lipids (astaxanthin, antioxidants, triglycerides, wax esters) can be used as they are for various applications, including feed in aquaculture, or the lipid classes can be further separated.
Compositions comprising high concentrations of omega-3 fatty acids are useful as pharmaceuticals for instance in the treatment of hypertriglyceridemia, dyslipidemia, hypercholesterolemia, heart failure, arterial fibrillation, coronary heart disease (CHD), vascular disease, atherosclerotic disease and related conditions, and the prevention or reduction of cardiovascular and vascular events.
Such compositions are K85EE or AGP (Pronova BioPharma ASA, Norway) which is the lipid composition in drug products like Omacor®, Lovaza™ and Seacor®. In this regard, in one embodiment, reference is made to U.S. Pat. No. 5,656,667 of Breivik et al. and possible fatty acids compositions disclosed therein.
In another embodiment, a pharmaceutical omega-3 fatty acid ethyl ester lipid composition comprises the following characteristics:
TABLE 4TestMinimum ValueMaximum ValueEPA EE1 (C20:5)430mg/g495 mg/gDHA EE1 (C22:6)347mg/g403 mg/gEPA EE1 and DHA EE1800mg/g880 mg/gTotal n-3 fatty acids90%(w/w)1EE is short for ethyl ester; i.e. EPA EE = EPA ethyl ester, and DHA EE = DHA ethyl ester.
Normally, K85EE and other compositions comprising very high concentrations of omega-3 fatty acids will have to be prepared by a combination of techniques, some of which work according to chain length (short path distillation, also called molecular distillation) and others which work according to degree of unsaturation (urea fractionation).
The processes short path distillation and molecular distillation are regarded as identical processes, the main issue being that the vacuum is high in order to keep the temperature low enough to avoid decomposition of the thermolabile polyunsaturated fatty acids. As used herein, the terms “thin-film distillation” and “falling-film distillation” are included in the term “short path distillation”.
We have now very surprisingly found that the krill lipids can be used to produce compositions like K85EE without using separation techniques that work according to degree of unsaturation (urea fractionation), or by only minor use of such techniques. By significantly reducing the amounts of urea that are needed for the production of K85EE, the yields are substantially increased and the cost of production is substantially reduced.